Novel Microbial Biomass Based Feed Products

ABSTRACT

Aquafeed, animal feed, and other food products, as well as nutritional and pharmaceutical compounds, chemicals and biomaterials are important commodities that can be produced at commercial scale by fermentation of microorganisms. The present invention provides a method for producing these valuable multi-carbon compounds from simple gas feedstocks, such as carbon dioxide, hydrogen and oxygen, by cultivating a consortium of microbial cells specially selected for this purpose in an aqueous culture medium. In addition to exploiting inexpensive feedstocks, such as waste industrial gas for this cultivation, the platform described herein also provides the advantage of removing carbon dioxide and other waste gases from industrial emissions, which would otherwise contribute to global climate change. Furthermore, the cultivation of a microbial consortium can provide highly nutritious components to a feed blend that might not be available from a monoculture.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/641,114 filed on Jul. 3, 2017 incorporated herein by reference. Thefollowing patent applications are also incorporated by reference herein:U.S. patent application Ser. No. 14/601,976 filed Jan. 21, 2015; U.S.Provisional Application 61/929,853 filed Jan. 21, 2014; U.S. ProvisionalPatent Application Ser. No. 62/358,048 filed Jul. 3, 2016; U.S. patentapplication Ser. No. 13/968,723 filed on Aug. 16, 2013; U.S. patentapplication Ser. No. 13/610,844 filed Sep. 11, 2012, now U.S. Pat. No.9,206,451 issued Dec. 8, 2015; U.S. Provisional Application 61/640,459filed Apr. 30, 2012; U.S. Provisional Application 61/533,672 filed Sep.12, 2011; U.S. patent application Ser. No. 12/726,980 filed Mar. 18,2010, now U.S. Pat. No. 8,518,566 issued Aug. 27, 2013; U.S. ProvisionalApplication 61/161,331 filed Mar. 18, 2009; U.S. patent application Ser.No. 13/034,596 filed Feb. 24, 2011; U.S. Provisional Application61/308,050 filed Feb. 25, 2010; U.S. Provisional Application 61/371,623filed Aug. 6, 2010; U.S. patent application Ser. No. 13/204,649 filedAug. 6, 2011; U.S. Provisional Application 61/371,623 filed Aug. 6,2010; U.S. patent application Ser. No. 13/841,704 filed Mar. 15, 2013;and U.S. Provisional Application 61/640,459 filed Apr. 30, 2012.

BACKGROUND Field of the Invention

The present invention is generally related to the fields of microbialfermentation and industrial biotechnology, including biological methods,processes and microorganisms for producing, via gas fermentation,nutritional products including high-protein food, food additives andother products from the gas streams which contain carbon and energy,which may be by-products, side products or products of variousindustrial processes.

Related Art

Gas feedstocks represent a vast resource of carbon and energy that canbe used to grow chemoautotrophic microorganisms for use in human andanimal nutritional products. Chemoautotrophic bacteria are capable ofcapturing and metabolizing carbon from inorganic sources, such as CO₂,CO, CH₄, as well as, in some cases, hydrogen (H₂) or methane (CH₄) as aprimary or additional source of energy. By converting inorganic carbonto organic carbon via metabolic carbon fixation, these microbes canserve as primary producers in natural environments. Many of thesechemoautotrophic species can be cultured on gas feedstocks inbioreactors for commercial production of biomass, which can be processedinto nutritional products such as animal feed, companion animal feed, oreven food for humans, and chemicals.

A microbial consortium is defined as two or more microbial speciesliving symbiotically. Microbial co-cultures. For the consortia describedas part of the present invention, chemoautotrophic microbes are employedin a system where the primary source of carbon and energy are bothsupplied as gases, such that the primary producers support a definedconsortium of bacterial species via mutualistic interactions. Theconsortium of various defined species in the present invention, whenharvested, can be used as a nutritional product. In order to make moreideal nutritional products, it is desirable to grow multiple differentspecies together, each of which provides different nutritionalproperties. The use of multiple microbes allows controlled modificationand customization of the nutritional composition of the final product.It is also possible to include one or more genetically modified strainswhich produce a key component, such as astaxanthin, a carotenoid whichis a key ingredient for aquaculture.

A formulation of bacteria that could increase the amounts of desirablefatty acids and amino acids in the final biomass product would thereforebe highly desirable.

An advantage of the present invention is that the dried biomass can beblended into an aquafeed or other nutritional product to replace thefishmeal that is normally harvested and used for feeding farmed fish andseafood, such as salmon, trout, tilapia, and shrimp. This will make amajor impact on reducing the stress on the world's fisheries (Pitcher &Cheung, 2013), which will not be able to keep up with the projecteddemand for fishmeal.

Another advantage of the present invention is that the composition ofthe consortium can be adjusted to fine-tune the nutritional compositionof the biomass product. This ability to make adjustments is advantageousbecause, for example, recent studies have shown that adapting the aminoacid composition of feed given to laboratory mice based on the animal'sown overall amino acid composition not only reduces the amount of feedneeded, but also improves the health of the. Also, aquaculture and otheranimal feeds and feed additives are altered by selecting the amounts ofvarious ingredients added to achieve desired amino acid profiles. Byadjusting the consortia, a number of application specific nutritionalproducts can be derived including for use by humans. Special consortiamay even be created for aid in treating medical conditions, dietarydeficiencies or to match growth stages of proposed consuming organism.

Nevertheless, bacteria comprising the genera Bacillus, Lactobacillus,Bifidobacterium and others, which are known to be of nutritional, and/ormedicinal and/or probiotic benefit are not capable of growing on gassubstrates, such as CO₂, H₂, CO and CH₄.

However, certain strains of chemoautotrophic microbes from the generaCupriavidus, Rhodobacter, Methylobacterium, Methylococcus,Rhodospirillum and Rhodopseudomonas, are known to grow on gaseoussubstrates, such as CO₂, H₂, CO and CH₄.

Photoautotrophic microbes are bacteria, cyanobacteria and algae that canfix carbon from CO₂ by utilizing light energy. However, certain speciesof photoautotrophic bacteria also exhibit chemoautotrophic metabolism,in that they are able to utilize hydrogen as an energy source to drivethe fixation of CO₂ without light. Examples of these are the generaRhodobacter, Rhodospirillum and Rhodopseudomonas. Many other suchspecies are known in the literature. C. necator has been shown to growfaster and more efficiently on CO2/H2 than typical acetogens that areused in some anaerobic gas fermentation.

Bacteria grown under chemoautotrophic conditions express differentamounts (and in some cases, different types) of proteins, enzymes,transporters, fats, oils, vitamins, co-factors and other biochemicals,than do those grown in traditional fermentation. Examples of this wouldbe cytochromes, quinones (e.g., coenzyme Q), RuBisCO(Ribulose-1,5-bisphosphate carboxylase oxygenase), as well as havingoverall different levels and ratios of amino acids. Because of this,bacteria grown chemoautotrophically have different nutritional profilesthan the same bacteria grown heterotrophically. These growth differencescan be advantages for providing additional vitamins, minerals,cofactors, etc. to the biomass product.

Bacteria grown chemoautrophically also have different secretoryactivity, which affects the content of the fermentation mix.Chemoautotrophic bacteria (and photoautotrophic bacteria grownchemoautotrophically) release chemicals into the growth medium, such asglycolates, polysaccharides, proteins, amino acids, fats, oils,hydrocarbons, nucleic acids, organic acids, polyhydroxyalkanoates,phasins, carotenoids, vitamins, gene transfer agents (GTA) and otherbiomolecules that can then be utilized by non-autotrophic bacteria andother heterotrophic microorganisms as growth substrates and growthregulators.

In an alternative embodiment, one or more of the strains in theconsortium may naturally, or be genetically modified in order to,produce a valuable small molecule (e.g., a specific fatty acid) or aprotein product (e.g., an enzyme, therapeutic protein, antibiotic,hormone, vitamin, precursor, antibody or vaccine). The present inventionprovides a means to produce such molecules using cheap gas feedstockseven if the host organism(s) cannot grow solely on gas.

In this application we describe an invention wherein a food or feedproduct with characteristics that provide nutritional, medical, and/ordietary benefits, and that comprises a consortium of chemoautotrophicmicrobes, photoautotrophic and non-chemoautotrophic microbes, isproduced by cultivating these microbes on gaseous substrates. Theconsortium creates an ecosystem in which the chemoautotrophic microbesform the base of a food chain on which the non-autotrophic microbes arecapable of growing. The goal of the gas-based fermentation processdescribed herein is to generate a biomass product that has enhancednutritional value compared to the biomass that the same chemoautotrophicmicrobes would produce if grown alone. A further economic benefit ofthis invention is to facilitate the growth of desired or beneficialmicrobes that otherwise could not utilize the inexpensive CO₂ or otherC1 feedstocks as a primary carbon source, and/or the hydrogen (or otherinorganic or C1 compounds) for energy.

A number of species of microbe are known to nutritionally beneficial, orto produce beneficial substances, or have probiotic properties which canbe significant components of a product which can be produced by thismethod. Examples of these are: Aspergillus niger, Aspergillus oryzae,Bacillus coagulans, Bacillus lentus, Bacillus licheniformis, Bacillusmegaterium, Bacillus pumilus, Bacillus subtilis, Bacteroidesamylophilus, Bacteroides capillosus, Bacteroides ruminocola, Bacteroidessuis, Bifidobacterium adolescentis, Bifidobacterium animalis,Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacteriumlactis, Bifidobacterium longum, Bifidobacterium thermophilum,Bifidobacterum breve, Lactobacillus acidophilus, Lactobacillus brevis,Lactobacillus bulgaricus, Lactobacillus casei, Lactobacilluscellobiosus, Lactobacillus curvatus, Lactobacillus delbruekii,Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillusjohnsonii, Lactobacillus lactis, Lactobacillus paracasei, Lactobacillusparafarraginis, Lactobacillus plantarum, Lactobacillus reuterii,Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillussporogenes, Lactococcus lactis, Leuconostoc mesenteroides, Pediococcusacidilactici, Pediococcus cerevisiae, Pediococcus pentosaceus,Propionibacterium shermanii, Propionibacterium freudenreichii,Saccharomyces boulardii, Saccharomyces cerevisiae, Streptococcuscremoris, Streptococcus diacetylactis, Streptococcus faecium,Streptococcus intermedius, Streptococcus lactis, Streptococcusthermophiles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bright field micrograph of a consortium grown on CO2, H2 andO2, which shows the multiple species of microbes, according to variousembodiments.

FIG. 2 is a schematic representation of a bioreactor according to anexemplary embodiment of the present invention, according to variousembodiments.

SUMMARY

In accordance with the present invention, products, materials,intermediates, and the like such as protein biomass and/or otherbiological products are produced from the waste gases of industrialprocesses by cultivating a microbial consortium. Such gases may includeCO₂, CO, CH₄, and H₂, thereby reducing environmental pollution while atthe same time saving energy and chemical feedstocks. Other trace gaseswhich may be present in the industrial effluent, such as H₂S or SO₂, canprovide additional nourishment to the autotrophic primary producerbacteria, and removal of such gases provides the added benefit ofremediating these toxic GHGs from the gas stream.

In accordance with an exemplary process of the present invention, thedesired components of the gas mixtures are introduced into a bioreactorcontaining one or more cultured strains of microbes that utilize thewaste gas components to produce a desired compound. The desired product(biomass, nutraceutical, protein, etc.) is recovered from the aqueousphase in a separate vessel or vessels, utilizing a suitable recoveryprocess for the compound produced. Examples of recovery processesinclude extraction, distillation or combinations thereof, or otherefficient recovery processes. The bacteria are removed from the aqueousphase and recycled to avoid toxicity and maintain high cellconcentrations, thus maximizing reaction rates. Cell separation, ifdesired, is accomplished by centrifugation, membranous ultrafiltration,or other techniques.

The principal object of the present invention is the provision of aprocess and/or a consortium of microorganisms for the production ofproducts, intermediates, materials, and the like such as biomass, feedingredients, proteins, vitamins, probiotics, natural antibiotics,organic acids, and the like from carbon dioxide, hydrogen, and oxygen.

Another object of the present invention is the provision of methods,microorganisms and apparatus for the production of items such asbiomass, feed ingredients, proteins, vitamins, probiotics, naturalantibiotics, and organic acids from the waste gas streams of industrialprocesses, such as brewing, bioethanol production, cement manufacturing,oil refining, and similar processes that generate waste CO₂ and/or H₂.

Yet another object of the present invention is the provision of amethod, microorganism and apparatus involving continuous gaseoussubstrate fermentation under aerobic conditions to accomplish theconversion of waste gas streams of certain industrial processes intouseful products such as biomass, feed ingredients, proteins, vitamins,probiotics, natural antibiotics, and organic acids.

Other objects and further scope of the applicability of the presentinvention will become apparent from the detailed description to follow,taken in conjunction with the accompanying drawings wherein like partsare designated by like reference numerals.

DETAILED DESCRIPTION

Microbial strains. C. necator has the advantage that it can grow veryrapidly and to high density on a mixture of H₂, CO₂, O₂, and it can becontinuously cultured for long periods of time without contamination.The same is true for C. basilensis, and for Methyloccocus capsulatuswhere CH4 is added or used in place of H2. Bacterial strains for theconsortium are selected for being both naturally-occurring (i.e.,non-GMO), generally recognized as safe (‘GRAS’) organisms, or because oftheir apparent beneficial qualities and apparent lack of negativecharacteristics, so that they will be broadly suitable for feed and foodprocessing, although GMO organisms designed for a specific purpose(e.g., metabolite production via an engineered pathway) can also beincluded, if desired. All strains for the invention described hereinwere obtained as pure, axenic type-cultures from culture collections.

Gas supply. The CO₂, H₂ and O₂ can be supplied from either flue gascollected from an industrial emitter (designated as ‘flue gas’) or frompure stocks of compressed gas obtained from a gas supplier (designatedas ‘lab gas’), from process gas produced by an industrial process, fromgasifier or pyrolysis output gas, syngas, from the manufacture ofcement, from a combustion process, or from any industrial, natural orother process which produces one or more of the desired gases. Forproduction of feed, food, nutraceuticals, biologicals, and the like, anindustrial source of waste CO₂ that is free of toxic elementalcontaminants (e.g., mercury) is preferred. Examples of such sourcesinclude CO₂ from breweries and bioethanol plants. Hydrogen can besupplied as part of the gas composition of pyrolysis gas, syngas, as anindustrial side product from activities such as propylene manufacture,as a component of a mixed gas stream from an oil refinery, or in gascreated by steam methane reformation (SMR) process), from compressedgas, or from electrolysis of water. Oxygen can be obtained fromatmospheric gas, as a product of electrolysis, or as a component ofindustrial by-product gas such as cement flue-gas. Embodiments of theinvention include methods for collecting and compressing flue gas at anindustrial site into transportable pressurized cylinders so thatsubstantial quantities of flue gas can be carried back to the laboratoryfor analysis and fermentation of the microorganisms. In the laboratory,the CO₂ and O₂ (derived from either lab gas or flue gas) are furtherdiluted approximately 5-fold with H₂ to supply the bacteria with afeedstock mixture that is optimized for growth. For a commercial-scaleoperation, the fermentation plant can be located near the gas productionsite, or the gas can be transported by vehicle or pipeline to thebiomass production site.

For injection into the fermenter, the gas supply was filtered through0.2 μm filters to remove particles and microorganisms. For small-scaleexperiments, compressed H₂, CO₂ and O₂ were each regulated to 20 psi.The gases were delivered to a flow proportioner, which sets the relativefraction of the gases, and a variable area flow meter to control themixture and flow rate into the fermenter. Gas flow was adjusted tobetween 0.2-1.2 VVM to supply adequate nutrients at each stage of thefermentation. The agitation rate was adjusted between 150-300 rpm toprovide thorough mixing.

Nutrient monitoring. The composition of the input and output gas can bemeasured and monitored to determine the gas uptake rates, the massbalances and the mass transfer efficiency for dissolution of the gasinto the solution and the biomass. Key nutrients (such as NH₄, PO₄ &SO₄), can also be monitored and replenished to prevent nutrientlimitations that might restrict bacterial growth.

Microbial inocula. The inocula for fermenter runs can be prepared inmany ways; each microbial strain may be grown separately, or two or moremay be grouped together in a single fermentation. Heterotrophic speciesare always grown up from pure cultures on heterotrophic medium that issuitable for propagating the particular species (or group of species)being grown. Chemoautotrophic species can be grown on gases, or, in somecases, on heterotrophic media. Photoautotrophic species may be grownusing light or heterotrophic media. Some photoautotrophic species arealso chemoautotrophic, and thus may be grown on gaseous substrates.Inoculating the bioreactor involves sterile addition of a culturecontaining one or more of the species into the bioreactor.

In some embodiments, all of the cultures for the consortium are added tothe bioreactor, in a short period of time, at the beginning of thefermentation procedure or run.

In some embodiments, chemoautotrophic microbes are added to thebioreactor at the beginning of the fermentation, and the inoculumcultures containing other species are added at later points.

In some embodiments, the timing of the addition, amount and density ofculture additions, and method of preparing inocula can be altered toaffect the qualities, composition, and/or value of the final product.

In some embodiments, additional inoculations of one or more strains usedin the consortia can be added at later times.

In embodiments of the invention, cultures were prepared by growing C.necator and the other chemoautotrophic species on H2/CO2/O2 to an OD₆₂₀^(˜)1 in small bottles of media equipped with gas fittings.Non-chemoautotrophic species were grown in liquid yeast-tryptone medium(YT medium), a well-known and commercially available medium. Thebioreactor was inoculated to OD^(˜)0.1. A ca. 5% inoculum is ideal. ThepH is controlled with 2N NH₄OH. The fermentation is run for up toseveral days, resulting in OD₆₂₀ of 1-100 or greater. The recoveredbiomass was analyzed for protein and lipid content and the compositionof each product. Proprietary strains, of some embodiments of theinvention, of C. necator and/or R. capsulatus, or other proprietarystrains of microbe, were sometimes used in addition to type strains.Embodiments of the invention include several strains of chemoautotrophicspecies that are adapted to flue gas and therefore tolerant to varioustoxic gas components, which can be included in the mix if complexindustrial flue gas is used as the feedstock. In some cases, additionalinoculations with one or more consortia strains were carried out a latertime points.

Microbe species commonly used in various embodiments for enablementinclude those shown in Table 1: The micrograph in FIG. 1 is from afermentation of all of these except B-3226.

TABLE 1 Strain Species Source B-3226 Rhodospirillum rubrum, (ARSNRRLType Strain) B-1727 Rhodobacter sphaeroides, (ARS NRRLType Strain)B-4276 Rhodopseudomonas palustris, (ARS NRRLType Strain) B-14308Bacillus megaterium, (ARS NRRLType Strain) B-356 Bacillus subtilis, (ARSNRRLType Strain) B-354 Bacillus subtilis, (ARS NRRLType Strain) B-14200Bacillus subtilis subspecies subtilis, (ARS NRRLType Strain) B-41406Bifidobacterium animalis subspecies animalis, (ARS NRRLType Strain)B-4495 Lactobacillus acidophilus, (ARS NRRLType Strain) B-1922Lactobacillus casei subspecies casei, (ARS NRRLType Strain) B-4383Cupriavidus necator, (ARS NRRLType Strain) B-14690 Cupriavidus necator,(ARS NRRLType Strain) Cupriavidus necator strain H16 (ATCC Type Strain)Rhodobacter capsulatus strain SB-1003 (ATCC Type Strain) OB213Rhodobacter capsulatus, Oakbio, Inc. Proprietary Strain OB311Cupriavidus necator, Oakbio, Inc. Proprietary Strain

Bioreactor Fermentation. A bioreactor for chemoautotrophic synthesis isused for enablement of this invention. Many types and designs ofbioreactor are suitable. The critical parts of a bioreactor forcultivation of the product discussed in this application are that therebe a vessel which is at least partially filled with liquid medium, inwhich the microbes are dispersed. The liquid comprises chemicalsrequired for growth of the microbes, examples of which are describedbelow. At least one port exists for introducing the gaseous substratesinto the liquid in the bioreactor. The vessel may have a headspace intowhich gases collect after traversing the fluid in the vessel. An exhaustport allows gases to exit the vessel. Additional ports are present asneeded for sensors, addition of liquids or chemicals and removal ofproduct, liquids, or samples for testing, as would be expected to befound on common bioreactors, which are well known in the field offermentation, cell culture and microbe cultivation. A minimal designbioreactor is shown in FIG. 2 . In various embodiments, the inventionhas been enabled in custom-built 250 ml, 1 L, and 4 L glass flask basedbioreactors and in a commercially manufactured New Brunswick ScientificBio Flo 4500 with the 4-gas handling option. Additional bioreactordesigns that can be used in conjunction with the present invention canbe found in U.S. patent application Ser. No. 13/204,649 filed on Aug. 6,2011 and entitled “Chemoautotrophic Bioreactor Systems and Methods ofUse” which is incorporated herein by reference. Bioreactors forembodiment of this invention can comprise one or more vessels and/ortowers or piping arrangements, and can comprise, for example, aContinuous Stirred Tank Reactor (CSTR), an Immobilized Cell Reactor(ICR), a Trickle Bed Reactor (TBR), a Bubble Column, a Gas LiftFermenter, a Static Mixer, a Fluidized Bed, an Up-flow or Down-flow, acontinuous, batch or loop reactor, or any other vessel or devicesuitable for maintaining suitable gas-liquid contact. In someembodiments, the bioreactor may comprise a first growth vessel and asecond chemoautotrophic synthesis vessel, while in other embodiments asingle vessel is used throughout both of the growth and synthesisstages.

In some embodiments, a gas recirculation system can be used to improvethe conversion efficiency, particularly during a continuous process, inorder to reduce the total gas requirement. Continuous harvesting of thecell mass is advantageous for a commercial production process, and canbe implemented through the continuous removal of cell broth and thecontinuous replenishing of medium, in order to maintain the culturevolume and cell density. Fermentations were run at a constant or variedtemperatures between 15 and 70 C, but the preferred temperature is 30 C.

Monitoring cell growth and species diversity. To monitor the progress ofcell growth and verify the species diversity of the culture, samples canbe periodically removed for analysis, or the bioreactor system cancomprise analytic equipment. In enabling the technology in someembodiments, characterization included microscopy of cell morphology, anexample of which is shown in FIG. 1 (from the completion of a run),which shows that the species diversity was maintained. Wet mounts of theculture were observed using brightfield microscopy with an Olympus BXresearch microscope equipped with an Amscope CCD camera. Micrographswere generated using the Amscope software for imaging and data storage.Species diversity can also be monitored and quantitated using methodswell known in the art, such as analysis of 16S rRNA genes, 23S rRNAgenes, or other genetic markers and phenotypic indicators (Jove) et al.,2016). Growth behavior was characterized by optical density (OD)measurements at 620 nm using an ICN TiterTek 96-well plate reader.Aliquots of each fermenter sample (200 ul) were measured in duplicate toplot the growth curves.

Carbon capture. Carbon capture from a new source of flue gas can beverified by performing headspace gas analysis, as well as growthexperiments that use the flue gas as the sole carbon source forbacterial biomass production. The dry weight of each culture can also bedetermined by centrifuging the culture, washing the pellet, drying thecells in a lyophilizer, and weighing the lyophilized cells.

Gas mixing. For hydrogen fermentations typically, the CO2 feedstock orraw flue gas was diluted with pure hydrogen with ratios of about 8:1 to1:1 (H2:CO2, v/v), resulting in a final CO2 concentration of about50%-1% or less. The O2 concentration is ideally 3-12%. For methanefermentations, typically the methane concentration is between 80% to 5%,CO2 is between 40%-1%, and oxygen is 50% to 5%. In either system, CO canbe up to 10%, and a variety of other gases may be present, includingsulfur oxides, nitrogen oxides, hydrogen sulfide, molecular nitrogen orother gases found in the gas source.

Culture medium. Many different mineral media recipes can be used, andvarying the media is one of the ways the characteristics of the finalproduct can be influenced. In various embodiments, typically a mineralsalts medium (modified from Repaske & Mayer, 1976) was used thatcontained no organic carbon or complex nutrients: Na2HPO4.2H2O 4.5 g/L,KH2PO4 1.5 g/L, NH4Cl 1.8 g/L, MgSO4.7H2O 0.11 g/L, NaHCO3 0.2 g/L,FeSO4.7H2O 12 mg/L, CaCl2.2H2O 10 mg/L, ZnSO4.7H2O 100 μg/L, MnCl2.4 H2O30 μg/L, H3BO3 300 μg/L, CoCl2.6H2O 200 μg/L, CuCl2.2H2O 10 μg/L,NiCl2.6H2O 20 μg/L, Na2MoO4.2H2O 30 μg/L.

Concentration and harvesting. Biomass product can be harvested throughmany methods, such as filtration, gravity separation, or other method,of which many are industrially practiced. Drying can be by spray drying,freeze drying, thermal drying, desiccation or many other methods, manyof which are currently practiced industrially.

Brief heat treatment is useful if the cells must be made non-viableprior to further processing. Due to the fact that lyophilization is moreenergy-intensive, it is more suitable for processing very high-valueproducts that require gentle processing. The dried material can beeasily blended with other ingredients to form a nutritious fish feedthat can replace aquafeed products that typically rely on fishmeal forprotein, fatty acids, and other nutrients. The amino acid composition ofthe dried material from a 30 L batch of cultivated consortium (Table 2)compares favorably to that of fishmeal (IAFMM Report, 1970)

In the below referenced demonstration, cell suspensions were removedfrom the fermenter via the sterile exit port. The supernatant can thenbe removed by centrifugation in a standard or process centrifuge at ca.4,000×g or greater to form a cell pellet. The cells are then washed in alow-salt buffer solution, and then re-pelleted. The final cell paste wasthen freeze dried to a powder using a commercial MTS lyophilizer.

TABLE 2 An example of the amino acid composition of a consortium. Asample of the consortia grown, in various embodiments, consisting ofchemoau- totrophs, photoautotrophs, and probiotic heterotrophs,cultivated at the 30 L scale in a New Brunswick Scientific BioFlo 4500Bioreactor with 4-gas input and control option. Amino acid compositionanalysis was conducted by NP Analytical Laboratories (St. Louis, MO).Amino Acid g/100 g Dried Biomass Aspartic Acid 4.49 Threonine 2.40Serine 1.79 Glutamic Acid 7.31 Proline 1.67 Glycine 2.15 Alanine 3.24Valine 3.34 Methionine 1.16 Isoleucine 2.60 Leucine 3.24 Tyrosine 1.71Phenylalanine 1.99 Histidine 0.930 Lysine 3.48 Arginine 2.45 Cysteine0.312 Tryptophan 0.519

PATENT CITATIONS

Publication Cited Patent Filing Date Date Applicant Title US Pat. No.9,267,158 B2 Jan. 7, 2016 Jul. 26, 2016 Intrexon Corp. Biologicalproduction of multi-carbon compounds from methane. US Pat. No. 7,579,163B2 Aug. 16, 2002 Aug. 25, 2009 Statoil Asa Method of fermentation USPat. No. 6,340,581 B1 Dec. 23, 1998 Jan. 22, 2002 BioengineeringBiological production of Resources, Inc. products from waste gases USPat. No. 9,206,451 B2 Sep. 11, 2012 Dec. 8, 2015 Oakbio, Inc.Chemoautotrophic conversion of carbon oxides in industrial waste tobiomass and chemical products EP 1419234 A1 Aug. 16, 2002 May 19, 2004Cockbain, Method of fermentation Julian, Norferm DA

FIG. 1 shows a bright field micrograph of a consortium grown on CO2, H2and O2, taken May 15, 2017. This picture shows a mix of chemoautotrophicand heterotrophic microbes grown with CO2 as a primary carbon source andH2 as a primary energy source. A short, thin rod 100, a medium-length,fat rod 110 (characteristic of C. necator), a long rod 120(characteristic of B. megaterium), and a coccus 130 are indicated byarrows.

FIG. 2 shows a schematic representation of a bioreactor 200 as oneexample of a bioreactor that is suitable for culturing a consortium ongas. Bioreactor 200 can comprise either a synthesis vessel for use inconjunction with a separate growth vessel, or can comprise a vesselsuitable for both of the growth and synthesis stages. In FIG. 2 ,bioreactor 200 includes a vessel 205 that in operation holds a quantityof a liquid medium 210 containing the chemoautotrophic micro-organismsand other microbes in culture. The bioreactor 200 also includes asubstrate port 215 through which a gaseous substrate 220 can beintroduced into the vessel 205 for introduction into the liquid medium210, a media inlet port 225 through which fresh media 230 can beintroduced into the vessel 205 for introduction into the liquid medium210, and a media outlet port 235 through which the medium 210 can beremoved, for example, to harvest biomass and/or chemical products. Thebioreactor 200 can also comprise a headspace 240 and a gas release valve245 to vent gases from the headspace 240. In some embodiments, the mediaoutlet port 235 and the media inlet port 225 are connected via a systemwhich harvests biomass 170 and reconditions the medium 210 forrecirculation. In some embodiments, the gas release valve 245 isattached to a system which re-circulates the gaseous substrate back tothe substrate port 215, and may make additions or subtractions tooptimize the gas composition.

Deposit of Biological Material

The following microbes have been deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, USA (ATCC):

TABLE 3 Microbe Designation ATCC No. Deposit Date Rhodobacter capsulatusOB-213 PTA-12049 Aug. 25, 2011

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of viable cultures for 30 years fromthe date of the deposit. The organisms will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Oakbio, Inc. and ATCC, which assures permanent and unrestrictedavailability of the progeny of the cultures to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 USC §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.12 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if the cultureson deposit should die or be lost or destroyed when cultivated undersuitable conditions, they will be promptly replaced on notification witha viable specimen of the same culture. Availability of the depositedstrain is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

What is claimed is:
 1. A method comprising: providing a consortium ofmicrobes within a system, the system including a fermentation vesselfilled with an aqueous medium comprising the consortium, thefermentation vessel having an input port into which gaseous substratesare introduced into the aqueous medium, the fermentation vessel furtherhaving an exhaust port through which gases exit the fermentation vessel,wherein the aqueous medium comprises inorganic anions and inorganiccations, the consortium including microbes from the genera Cupriavidus,Rhodococcus, or Methylococcus, microbes from the genera Rhodobacter,Rhodospirillum, Rhodopseudomonas, or Arthrospira, and microbes from thegenera of Bacillus, Bacteroides, Bifidobacterium, Lactobacillus,Lactococcus, Leuconostoc, Acidilactici, Pediococcus, Propionibacterium,or Streptococcus; introducing a first gaseous substrate into the inputport, the first gaseous substrate including carbon dioxide; introducinga second gaseous substrate into the aqueous medium, the second gaseoussubstrate including predominantly hydrogen gas (H₂), wherein either ofthe first or second substrates additionally includes oxygen gas (O₂);and harvesting cells of the microbes from the system to generate abiomass product.
 2. The method of claim 1, wherein the microbes from thegenera Cupriavidus, Rhodococcus, or Methylococcus include one or more ofCupriavidus necator, Cupriavidus basilensis, and Methylococcuscapsulatus; the microbes from the genera Rhodobacter, Rhodospirillum,Rhodopseudomonas, or Arthrospira further include one or more ofRhodospirillum rubrum, Rhodobacter sphaeroides, and Rhodopseudomonaspalustris; and the microbes from the genera of Bacillus, Bacteroides,Bifidobacterium, Lactobacillus, Lactococcus, Leuconostoc, Acidilactici,Pediococcus, Propionibacterium, or Streptococcus include one or more ofAspergillus niger, Aspergillus oryzae, Bacillus coagulans, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus subtilis, Bacteroides amylophilus, Bacteroides capillosus,Bacteroides ruminocola, Bacteroides suis, Bifidobacterium adolescentis,Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacteriuminfantis, Bifidobacterium lactis, Bifidobacterium longum,Bifidobacterium thermophilum, Bifidobacterum breve, Lactobacillusacidophilus, Lactobacillus brevis, Lactobacillus bulgaricus,Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus curvatus,Lactobacillus delbruekii, Lactobacillus fermentum, Lactobacillushelveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillusparacasei, Lactobacillus parafarraginis, Lactobacillus plantarum,Lactobacillus reuterii, Lactobacillus rhamnosus, Lactobacillussalivarius, Lactobacillus sporogenes, Lactococcus lactis, Leuconostocmesenteroides, Pediococcus acidilactici, Pediococcus cerevisiae,Pediococcus pentosaceus, Propionibacterium shermanii, Propionibacteriumfreudenreichii, Saccharomyces boulardii, Saccharomyces cerevisiae,Streptococcus cremoris, Streptococcus diacetylactis, Streptococcusfaecium, Streptococcus intermedius, Streptococcus lactis, Streptococcusthermophiles.
 3. The method of claim 1, wherein the microbes from thegenera Cupriavidus, Rhodococcus, or Methylococcus comprise Cupriavidusnecator.
 4. The method of claim 1, wherein the microbes from the generaof Bacillus, Bacteroides, Bifidobacterium, Lactobacillus, Lactococcus,Leuconostoc, Acidilactici, Pediococcus, Propionibacterium, orStreptococcus comprise Bifidobacterium animalis.
 5. The method of claim1, wherein the microbes from the genera of Bacillus, Bacteroides,Bifidobacterium, Lactobacillus, Lactococcus, Leuconostoc, Acidilactici,Pediococcus, Propionibacterium, or Streptococcus comprise Lactobacillusparafarraginis.
 6. The method of claim 1, wherein the microbes from thegenera of Bacillus, Bacteroides, Bifidobacterium, Lactobacillus,Lactococcus, Leuconostoc, Acidilactici, Pediococcus, Propionibacterium,or Streptococcus include Lactobacillus acidophilus or Lactobacilluscasei.
 7. The method of claim 1, wherein the microbes from the genera ofBacillus, Bacteroides, Bifidobacterium, Lactobacillus, Lactococcus,Leuconostoc, Acidilactici, Pediococcus, Propionibacterium, orStreptococcus include Bacillus megaterium.
 8. The method of claim 1,wherein the microbes from the genera of Bacillus, Bacteroides,Bifidobacterium, Lactobacillus, Lactococcus, Leuconostoc, Acidilactici,Pediococcus, Propionibacterium, or Streptococcus include Bacillussubtilis.
 9. The method of claim 1, wherein providing the consortiumincludes adding the microbes from the genera Cupriavidus, Rhodococcus,or Methylococcus into the fermentation vessel and then adding themicrobes from the genera Rhodobacter, Rhodospirillum, Rhodopseudomonas,or Arthrospira and the microbes from the genera of Bacillus,Bacteroides, Bifidobacterium, Lactobacillus, Lactococcus, Leuconostoc,Acidilactici, Pediococcus, Propionibacterium, or Streptococcus into thefermentation vessel.
 10. The method of claim 1, wherein providing theconsortium includes adding the microbes into the fermentation vesselsimultaneously.
 11. The method of claim 1, wherein providing theconsortium includes adding the microbes into the fermentation vessel atdifferent times.
 12. The method of claim 1, wherein a concentration ofsingle-carbon atom molecules in the first gaseous substrate and aconcentration of hydrogen gas in the second gaseous substrate are bothhigher concentrations than are found in the ambient atmosphere.
 13. Themethod of claim 1, wherein the single-carbon atom molecules comprise CO₂and the first gaseous substrate includes a concentration of CO₂ and aconcentration of CH₄, wherein the second substrate includes aconcentration of hydrogen gas, and wherein all three concentrations arehigher concentrations than are found in the ambient atmosphere.
 14. Themethod of claim 1, wherein the single-carbon atom molecules comprise CO,CO₂, and CH₄, and the first gaseous substrate includes a concentrationof CO₂, a concentration of CH₄, and a concentration of CO, wherein thesecond substrate includes a concentration of hydrogen gas, and whereinall four concentrations are higher concentrations than are found in theambient atmosphere.
 15. The method of claim 1, wherein the single-carbonatom molecules comprise CO and CH₄ and the first gaseous substrateincludes a concentration of CO and a concentration of CH₄, wherein thesecond substrate includes a concentration of hydrogen gas, and whereinall three concentrations are higher concentrations than are found in theambient atmosphere.
 16. The method of claim 1, wherein the single-carbonatom molecules comprise CH₄, CO, and CO₂.
 17. The method of claim 1,wherein the single-carbon atom molecules comprise CO and CH₄ and thefirst gaseous substrate includes a concentration of CO and aconcentration of CH₄, and wherein both concentrations are higherconcentrations than are found in the ambient atmosphere.
 18. The methodof claim 1, wherein the inorganic anions comprise phosphate, nitrate,sulfate, carbonate, or ammonium.
 19. The method of claim 1, wherein theinorganic cations comprise iron, nickel, calcium, magnesium, manganese,or cobalt.
 20. The method of claim 1, wherein one of the microbes of theconsortium has been genetically modified.
 21. The method of claim 20,wherein one of the genetically modified microbes of the consortiumproduces a carotenoid.
 22. The method of claim 20, wherein one of thegenetically modified microbes of the consortium overproduces a vitamin.23. The method of claim 20, wherein one of the genetically modifiedmicrobes of the consortium overproduces a protein.
 24. The method ofclaim 20, wherein one of the genetically modified microbes of theconsortium produces astaxanthin.
 25. The method of claim 1, wherein themicrobes from the genera Rhodobacter, Rhodospirillum, Rhodopseudomonas,or Arthrospira include any of Rhodobacter sphaeroides andRhodopseudomonas palustris, the microbes from the genera of Bacillus,Bacteroides, Bifidobacterium, Lactobacillus, Lactococcus, Leuconostoc,Acidilactici, Pediococcus, Propionibacterium, or Streptococcus includeany of Bacillus megaterium, Bacillus subtilis, Bifidobacterium animalis,Lactobacillus acidophilus, and Lactobacillus casei, and the microbesfrom the genera Cupriavidus, Rhodococcus, or Methylococcus include anyof Cupriavidus necator, Cupriavidus basilensis, and Methylococcuscapsulatus.
 26. The method of claim 1, wherein the microbes from thegenera Cupriavidus, Rhodococcus, or Methylococcus include microbes fromthe species Cupriavidus necator.